FOODCROPS.VN. R.K. Singh .Breeding for salt tolerance in rice

1. R.K.Singh
R.K. Singh .Breeding for Salt Tolerance in Rice
R.K. Singh
PBGB, IRRI

2. R.K.Singh
 Extent of the problem and
management options
 Reason of Limited Success
 Plant adaptation – salt
tolerant mechanisms
 Morphological symptoms
 Basic concepts (genotype vs.
phenotype and heritability)
 Genetic Studies
 Screening techniques
 Breeding strategy
 Physiological mechanisms
 Molecular mapping
 Varietal development
 NRM approaches
Outlines of the Lecture

3. R.K.Singh
EXTENT OF SALT-AFFECTED SOILS
World’s Total area
12.78 b ha
340 x 106
ha (Ponamperuma, 1984)
954 x 106
ha ( Massoud, 1974)
10% area ~ 1.2 b ha (Tanji, 1991)
FAO Database
397 x 106
ha (3.1%) – Saline soils
434 x 106
ha (3.4 %)– Sodic Soils
Asia, Pacific andAsia, Pacific and
Australia (M ha)Australia (M ha)
195 249
Source : FAO database
Total : 444 M ha

4. R.K.Singh
What are the salt-affected soils ?

5. R.K.Singh
How to Manage the Salt-affected Areas ?
1. Environment
modifying approach :
Change the
environment for the
normal growth of plants
2. Crop based
approach : Select or
develop crop variety
which can withstand
the salt stress
Do we need
ST
cultivars ?
Rice has
enormous
variability

6. R.K.Singh
Management of the Salt-affected Soils
3. Hybrid Approach
It is the combination of
environment modifying
and plant based approach.
Advantages:
• More viable
• Highly productive
• Low resource cost
Local variety without gypsum
Salt tolerant rice variety,
CSR13, with 25% Gypsum

7. R.K.Singh
Reasons of Limited Success
 Salt stress seldom happen in isolation
 Harsh, highly variable environment, large G/E
 Lack of efficient / precise screening procedure
 Lack of mechanistic understanding
 Low priority and less number of researchers involved

8. R.K.Singh
Salt Stresses and Associated Complexities
S
A
L
T
S
T
R
E
S
S
E
S
Acid
SO4
Peat
S
A
L
I
N
E
ALKALINE
INLAND
SALINE
(P, Zn)
(P, Zn)
(P, Zn)
(P, Zn)
(Fe)
Fe, Al
tox
Fe, H2S
tox
Al, Organic
Acids tox
(P & Zn)
R
A
I
N
F
E
D
Sub-
merged
Deep-
water
Drought
Irrigated
G
r
a
I
n
Q
u
a
l
i
t
y
(Source: Glenn B. Gregorio)

9. R.K.Singh
Breeding
for
Salt tolerance + High productivity
• Na+
Exclusion
• Tissue tolerance
• K+
uptake
• Partitioning etc.
All are quantitative trait
Quantitative trait
Single trait

10. R.K.Singh
1. Restricting the entry of toxic
ions at root level - Exclusion
2. Transporting the toxic ions to
stem, leaf sheath or older leaves –
plant level compartmentation
4. Sequestration of the toxic ions
to vacuole or cell wall – cell level
compartmentation
3. Excretion of salt through salt
glands, salt-hairs or bladders – in
most halophytes
Predominant salt-tolerance mechanisms operating in plant
Na+
Cl-

11. R.K.Singh
Physiology: traits associated with
salinity tolerance
Regulation of uptake
Compartmentation
In old tissue
Upregualtion of
antioxidants
Vigorous growth
Responsive
stomata
[Na+
]
OsmoprotectantsAOSS
K+
AtNHX1
H+
Na+
Vacuolar Na+
/H+
SOS1
Na+
H+
Plasma Na+
/H+
AVP1
H+
PPiase
Compartmentation within tissue
(tissue tolerance)
Protective
metabolites
Polyamines,
dehydrins,
glyoxalates
Earliness
Source : A.M. Ismail

12. R.K.Singh
Morphological Symptoms
 White leaf tip followed by tip burning (salinity)
 Leaf browning & death (sodicity)
 Stunted plant growth
 Low tillering
 Spikelet sterility
 Low harvest index
 Less florets per panicle
 Less 1000 grain weight
 Low grain yield
 Change in flowering duration
 Leaf rolling
 White leaf blotches
 Poor root growth
 Patchy growth in field
Manifestation of Salt Stress

13. R.K.Singh
First symptom
“Leaf tip
burning”
“Leaf tip burning
extends toward
base through
Lamina”
“Ultimate death
of leaf – always
from oldest to
youngest”
Salinity symptoms at the vegetative stage

14. R.K.Singh
Effect of salinity at Reproductive stage – Spikelet Sterility

15. R.K.Singh
Effect of salinity at reproductive stage – papery sterile spikelets

16. R.K.Singh
Physiological & Biochemical
 High Na+
transport to shoot
 Preferential accumulation of Na in older leaves
 High Cl-
uptake
 Lower K+
uptake
 Lower fresh and dry weight of shoot and roots
 Low P and Zn uptake
 Increase of non-toxic organic compatible solutes
 Increase in Polyamine levels
Manifestation of Salt Stress
Screening parameters ?

17. R.K.Singh
Which is the most reliable stage for screening ?
Association between
Correlation Coeff.
Glasshouse
studies
Field studies
Veg. stage tolerance vs. Grain yield
Rep. stage tolerance vs. Grain yield
Veg. stage vs. Rep. stage tolerance
- 0.58ns
- 0.97**
0.59ns
- 0.022ns
- 0.82**
0.34ns
Vegetative vs. Reproductive stage salt tolerance

18. R.K.Singh
Chaffy panicles
Papery florets
IR66946-3R-178-1-1

19. R.K.Singh
Number of gene(s) responsible for a trait (n) / Genotypic classes
1 2 5 10
F2
F3
F4
F5
F6
1:2:1
3:2:3
7:2:7
15:2:15
31:2:31
1:2:1:2:4:2:1:2:1
3:2:3:6:4:6:3:2:3
7:2:7:14:4:14:7:2:7
15:2:15:30:4:30:15:2:15
31:2:31:62:4:62:31:2:31
243
classes
59,049
classes
P* 1/4 1/16 1/ 1,024 1/ 1,084,576
*: P is the probability of getting the desired homozygote at all the loci in smallest perfect
population in F2 (1/4n
)
Trait A = 5 loci -- Desired recombinant – 1/1,024
Trait B = 10 loci -- 1/1,084,576
Prob. of getting both desired one in one background = 1/1,024 x 1/1,084,576
= 1/ 1,110,605,824 (> 1b)
Probability of getting the desirable genotype
Why Recurrent selection – mating of the selected individuals ?

20. R.K.Singh
Precision vs. Resources
Precision
l
r
y
Resources
No. of
low
more
Very High
Since the salinity is highly variable in soil due to the dynamic state
of soluble salts hence one should go for more blocks at different
locations over the years (judiciously compromising the resources)
for the precise estimates

21. R.K.Singh
Based on reproductive stage tolerance
Bas. 370 / CSR10
Bas. 370 / CSR11 Pak. Bas. / CSR10
Controlled by numerous minor genes as
revealed by the normal distribution curve
with few major genes (skewness)
SALINITY
Substituted
Genetics of Salt Tolerance
Inheritance Pattern

22. R.K.Singh
Inheritance for sodicity tolerance
Similar results (based on the same
crosses
Genetics of Salt Tolerance
P1
XP2 F1
X
F1 X
P1

23. R.K.Singh
SV df Mean Square
GCA
SCA
Error
5
15
40
15.36*
3.71*
0.22
0.238*
0.066*
0.012
0.100*
0.033*
0.006
Genetics of Salt tolerance
Gene Action (based on 6x6 diallel)

24. R.K.Singh
Seedling stage tolerance:In 20 days can classify the
tolerance level
Screening Techniques Standardised

25. R.K.Singh
Screening technique standardised
KR1-24
Tolerantcheck
IR66946-3R
IR29(sens
Muskan41
Cheriviruppu
IR66946-3R
IR29
NB: Instead of Pokkali, now IR 66946-3R-178-1-1 is being used as tolerant check
which is derived from IR29 / Pokkali cross. It is semi-tall, photoinsensitive and highly
salt tolerant

26. R.K.Singh
Normal
Saline
1 2 3
1 2 3
FL478 / IR29 FL478 / IR29 FL478 /
IR29
FL478 / IR29 FL478 / IR29 FL478 / IR29
Performance of 1 mo-old FL478 (tolerant line) and IR29 (susceptible variety) rice seedlings
under normal and saline (14d EC12 then 14d EC18) conditions using SNAP and nutrient
solutions: (1) 100% SNAP solution in tap water, (2) 75% SNAP solution in tap water, and (3)
nutrient solution in distilled water.
(Source: Dante Adorada)

27. R.K.Singh
Comparison between 28-day old rice seedling grown for 21 days in SNAP solution (Simple
Nutrient Addition Program) with (a) 100% nitrate and (b) 90% nitrate & 10% ammonium in
their composition.
(Source: Dante Adorada)

28. R.K.Singh
Phenotyping for the Adult Plant Salinity Tolerance
Microplots with controlled salinity and sodicity
Sodic Soil Environment
Saline Soil Environment
(Rain shelter)
Automatic Circulatory Solution Culture System

29. R.K.Singh
 Nais the most notorious element causing salt related
problems in plants
 Its higher uptake hinders the metabolic activities in plants
 Plants try to resist this element using various physiological
mechanisms
• Na+
exclusion,
• Tissue Tolerance
• Higher K+
uptake to counter Na
• Compartmention (Preferential accumulation of Na+
in stem,
leaf sheath, older leaves etc.)
• Early vigour
• …… Many more
Salinity Tolerance in Rice

30. R.K.Singh
Breeding Strategy
Identification of the genotypes based on the inherent
physiological mechanism (Na exclusion, K uptake, Tissue
tolerance and high initial vigor etc.) responsible for salinity
tolerance
Inter-mating of the genotypes with high degree of expression
of the contrasting salinity tolerance mechanism
Identifying / screening of the recombinants for pooling/
pyramiding of the mechanisms

31. R.K.Singh
Identify the donors for predominant physiological mechanisms
responsible for salt tolerance
• Na+
exclusion,
• Tissue Tolerance
• K+
uptake,
• Preferential accumulation of Na+
in stem, leaf sheath, older
leaves etc.
• Early vigour
However, none of the rice variety posses all the possible positive
mechanism conferring salinity tolerance.
Breeding Strategy

32. R.K.Singh
Grouping of the rice varieties on the basis of
Na accumulation per day
B a s .3 7 0 , C S R 1 0 , C S R 1 9
M I-4 8 , B a s .3 8 5 ,
C S R 1 8 , P R 1 0 8
L o w
< 0 .1 m m o l/g
C S R 1 1 , IR 3 6 , H B C 1 9 , C S R 2 0 , A D T 3 6
H K R 1 2 8 , C S R 1 , J a y a , C S R 1 3 , A c h h i
S u k h v e l, IR 4 2 , IR 2 4 , M a jh e ra 7 , M a n g la
S L R 5 1 2 1 4 , P ra s a d , V a n d n a , S a liv a h n a
M e d iu m
0 .1 - 0 .4 9 9 m m o l/g
S R 2 6 B , C S R 2 1 , IR 4 6 3 0 , P o k k a li, T -2 3
G R 1 1 , P a n v e l-2 , In d ra s a n , IR 5 8 , R P 1 4 4
H a th w a n , C a rp s C la rk , S w a rn d h a n , R a v i
U d a y a , T -2 1 , M a jh e ra -3 , B a rk a t, M K 4 7 -2 2
H ig h
> 0 .5 m m o l
N a a c c u m u la tio n p e r d a y
(m m o l/g d w t)

33. R.K.Singh
C S R 2 1 , IR 4 6 3 0 , H a th w a n , S w a rn d h a n
In d ra s a n , A c h h i, M u s k a n , U d a y a
R P 1 4 4 , V K L -3 9 , C a rp s C la r k
H ig h
> 0 .4 m m o l/g
C S R 1 1 , IR 3 6 , C S R 2 0 , P a n v e l-2 3 , B C 1
R a v i, S a liv a h a n a , H a s a n S a ra i, B a rk a t
P R 1 0 6 , IR 5 8 , IR 2 4 , M a jh e ra -3 , A D T 3 6
S L R 5 1 2 1 4 , IR 4 2 , M K 4 7 -2 2 , T -2 3 , M a n g la
M e d iu m
0 .2 - 0 .4 m m o l/g
S R 2 6 B , H B C 1 9 , C S R 1 , P o k k a li, T -2 3
G R 1 1 , P R 1 0 8 , C S R 1 0 , C S R 1 8 , C S R 1 9
J a y a , H K R 1 2 8 , M I-4 8 , B a s .3 7 0 , B a s .3 8 5
M a jh e ra -7 , P r a s a d , V a n d n a , V ik ra m a ry a
L o w
< 0 .2 m m o l
K a c c u m u la tio n p e r d a y
(m m o l/g d w t)
Grouping of the rice varieties on the basis of
K accumulation per day

34. R.K.Singh

35. R.K.Singh

36. R.K.Singh
Rice variety A
Good excluder
+
poor tissue tolerance
Rice variety B
Poor control at root level
+
High tissue tolerance
Dustbin
Garbage
Na+
Rice variety C
Good excluder
+
High tissue tolerance
K+

37. R.K.Singh
An Ideal High Yielding Salinity Tolerant Variety
 Highly tissue tolerant
 Good Excluder- Minimum per day uptake of Na+
 High uptake of K+
per day
 Low Cl-
uptake
 Low Na+
/ K+
ratio
 Good initial vigour
 Agronomically superior with high yield potential (plant type
+ grain quality)

38. R.K.Singh
Breeding Strategy
Grouping of the genotypes based on the inherent
physiological mechanism responsible for salinity
tolerance
Inter-mating of the genotypes with high degree of
expression of the contrasting salinity tolerance
mechanism
Identifying / screening of the recombinants for pooling/
pyramiding of the mechanisms - MAS

39. R.K.Singh
RM283
R844
S2139
RM23
RM140
RM113
S1715
S13994
RM9
RM5
C1456
RM237
RM246
0.0
27.4
28.4
40.0
64.9
66.2
71.2
75.3
77.2
91.9
98.2
99.1
103.1
119.5
123.5
129.9
A
C52903S
C1733S
R2374B
C52903S
C1733S
R2374B
RM283
R844
S2139
RM23
RM140
RM113
S1715
S13994
RM9
RM5
C1456
RM237
RM246
0.0
27.4
28.4
40.0
75.3
77.2
91.9
98.2
99.1
103.1
119.5
123.5
129.9
RM283
R844
S2139
RM23
RM140
RM113
S1715
S13994
RM9
RM5
C1456
RM237
RM246
0.0
27.4
28.4
40.0
75.3
77.2
91.9
98.2
99.1
103.1
119.5
123.5
129.9
AP3206
RM3412
CP03970
RM8094
RM493
CP6224
RM140
0.0
1.0
1.8
1.9
1.2
1.3
Short arm of chromosome 1
Progress of Saltot locus
• Saturated map of the
Chromosome 1
(Saltol segment) is
developed
• Closely linked
markers linked to
the saltol locus
identified
• MAS is being
validated in 3
breeding populations
60.6
(Source: Glenn B. Gregorio)

40. R.K.Singh
LOD threshold
RM140
CP6224
RM493
RM8094
CP03970
RM3412
CP010136
AP3206
0.0
b
a
Chromosome location of associated QTL of
Salinity tolerance trait
2.5

41. R.K.Singh
• preprotein translocase, SecA subunit
• Sec23/Sec24 trunk domain, putative
• Ser Thr Kc
• Protein kinase domain
• S-adenosylmethionine synthetase
• chloroplast membrane protein
•Cold shock protein
• secretory peroxidase
• CBL-interacting protein kinase 19
• Peroxidase, putative
• Cell wall protein type (Extensin,
Hydorxyproline rich, glycine rich)
• phospholipid/glycerol
acyltransferase –like
• Mitochondrial carrier
protein, putative
• GDSL-like
Lipase/Acylhydrolase,
putative
• organic cation transporter
• major facilitator
superfamily protein
•Cell wall protein type
(Extensin,Hydorxyproline
rich, glycine rich)
• CP12 domain, putative
• Stress-inducible membrane pore protein
• Zinc finger, C3HC4 type (RING finger),
putative
• Universal stress protein family
• Cation-chloride co-transporter
• Receptor like protein kinase
• Myb-like DNA-binding domain, putative
• Peroxidase, putative
• Cell wall protein type
(Extensin,Hydorxyproline rich, glycine
rich)
• Cation transporter
• Phospholipase D. Active site motif,
putative
• Protein kinase domain, putative
• Dual specificity phosphatase, catalytic
domain, putative
• Pectinemethyesterase/invertase inhibitor
• Pectinesterase
Rice Chromosome 1
60.6 60.9 62.5 64.9 65.4 66.2 67.6 67.9
cM
65.8
Saltol region ( Major QTL K+
/Na+ratio )
(Source: Ellen Tumimbang)

42. R.K.Singh
11.9 Mb 12.13 Mb
12.11Mb 12.27Mb
12.25Mb 12.40Mb
12.0Mb 12.27 Mb
preprotein
translocase,
SecA subunit
Sec23/Sec2
4 trunk
WD40
Ser Thr
Kc
Receptor
like kinase
SAM
synthetase
cold
shock
protein
chloroplast
membrane
protein
secretory
peroxidase
CBL-interacting
protein kinase 19
Peroxidase,
putative
S_Tkc;
WD40
0.27 Mb
SALtol Region ( Major QTL
K+
/Na+)
(~40 genes)
11.10Mb 12.7Mb
60.6 60.9 62.5 64.9 65.4 66.2 67.6 67.9
cM
65.8
Chromosome 1 of Rice
B1135C02
OSJNBa0011P19
P0426D06
B1153f04
(Source: Ellen Tumimbang)

43. R.K.Singh
List of genes that are located in the region of QTL and up-
regulated by high salinity in rice
Gene name
Insertion
lines
Clone ID
full length
cDNA
Rice 60k chip data
under high salinity
(fold-induction)
References
0.5 h 2 h 6 h
Pectinesterase 1B-23740,
1B-23741
CG408589
Ak105998 1.1 3.3 4.9
Ser/thr kinase AK065231 2.3 2.7 Guo et al., 2001
Phospholipase D 1515 AK120868 3.5 2.6 Kacperska, 2004
Zhu, 2002
SecA/protein
transport factor
CL520490
CL520492
AK070488 3.1 1.5
Peroxidase AK099187 2.6 3.05 Pastori and Foyer, 2002
Sottosanto et al., 2004
Alkaline Invertase AK120720 4.0 2.2 4.2
Unknown cDNA AK099887 0.37 1.6 2.4
(Source: Ellen Tumimbang)

44. R.K.Singh
• Putative SecA-type
chloroplast protein
transport factor
• Serine/threonine kinase
• Peroxidase
• Pectinesterase
• Phospholipase D. Active site
motif -- putative
The position of the candidate genes in chromosome 1
60.6 60.9 62.5 64.9 65.4 66.2 67.6 67.9
cM
65.8
Saltol region ( Major QTL K+
-Na+ratio )
Plant
neutral/alkaline
invertase
(Source: Ellen Tumimbang)

45. R.K.Singh
Mapping Salinity Tolerance Genes
at Reproductive Stage
QTLs for salinity tolerance genes at seedling
stage are different from reproductive stage
• Seedling stage tolerance in chrom 1.
• Reproductive stage tolerance in chrom 3, 4, 7,
and 9
Dr. Mirza M. Islam
Ph.D.

46. R.K.Singh
Salt tolerant rice varieties developed by IRRI and released
in Philippines
IRRI 112 - PSBRc48 (Hagonoy)
IRRI 113 - PSBRc50 (Bicol)
IRRI 124 - PSBRc84 (Sipocot)
IRRI 125 - PSBRc86 (Matnog)
IRRI 126 - PSBRc88 (Naga)
IRRI 128 - NSICRc106
Other salt-tolerant rice varieties
CSR10, CSR13, CSR23, CSR27, CSR30, CSR36 and Lunishree, Vytilla 1,
Vytilla 2, Vytilla 3, Vytilla 4, Panvel 1, Panvel 2, Sumati, Usar dhan 1, 2
& 3 (India); BRRI dhan 40, BRRI dhan 41 (Bangladesh); OM2717,
OM2517, OM3242 (Vietnam)

47. R.K.Singh

48. R.K.Singh
Realization of the Genetic Potential
 Promote the
Interdisciplinary
IRRI-NARS
collaborative
research, based
on CNRM
technology and
its validation in
the farmers
participatory
mode

49. R.K.Singh
Progress in salinity research
 = completed, = fast track, = not available
/available /on-going
Saline Sodic Zn-def Acid
Donor    
Screening
technique
   
   
Mechanism    
Genetics  ?  
MAS
development
   
Elite lines    ?
Lab.
Field

50. R.K.SinghThanks for Your Kind Attention
Glenn B. Gregorio Rafiqul Islam Mirza M. Islam Jong C. Ko
R K Singh Andy Sajise Ghasem M. Nejad Glenn Alejar
Adorada Dante Venus Elec Swe Thein Midie
Rhulyx Mendoza Jean Melgar Lorelie Ramos Venessa
Ellen Tumimbang Jaarmi Orly Kelvin
Rollin De Ocampo Angelito Francisco

51. R.K.Singh
Please feel free to contact any time
r.k.singh@cgiar.org